Identification and Functional Characterization of a Soybean (Glycine max) Thioesterase that Acts on Intermediates of Fatty Acid Biosynthesis.

Huong Thi Diem Tran,Nhan Trong Le,Thuong Thi Hong Nguyen,Vy Le Uyen Khuat

doi:10.3390/plants8100397

Huong Thi Diem Tran, Nhan Trong Le + Show 2 more

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https://doi.org/10.3390/plants8100397

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Abstract

(1) Background: Plants possess many acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) with unique specificity. One such TE is methylketone synthase 2 (MKS2), an enzyme with a single-hotdog-fold structure found in several tomato species that hydrolyzes 3-ketoacyl-ACPs to give free 3-ketoacids. (2) Methods: In this study, we identified and characterized a tomato MKS2 homolog gene, namely, GmMKS2, in the genome of soybean (Glycine max). (3) Results: GmMKS2 underwent alternative splicing to produce three alternative transcripts, but only one encodes a protein with thioesterase activity when recombinantly expressed in Escherichia coli. Heterologous expression of the main transcript of GmMKS2, GmMKS2-X2, in E. coli generated various types of fatty acids, including 3-ketoacids—with 3-ketotetradecenoic acid (14:1) being the most abundant—cis-Δ5-dodecanoic acid, and 3-hydroxyacids, suggesting that GmMKS2 acts as an acyl-ACP thioesterase. In plants, the GmMKS2-X2 transcript level was found to be higher in the roots compared to other examined organs. In silico analysis revealed that there is a substantial enrichment of putative cis-regulatory elements related to disease-resistance responses and abiotic stress responses in the promoter of this gene. (4) Conclusions: GmMKS2 showed broad substrate specificities toward a wide range of acyl-ACPs that varied in terms of chain length, oxidation state, and saturation degree. Our results suggest that GmMKS2 might have a stress-related physiological function in G. max.

Highlights

In plants, the formation of acyl carrier protein (ACP) thioesters of acyl groups containing between2 and 16 carbon atoms occurs in fatty acid (FA) biosynthesis and is catalyzed by a set of fatty acid synthase (FAS) enzymes highly conserved across various species [1,2]
This search identified only one methylketone synthase 2 (MKS2)-like gene sequence encoding a protein with homology to ShMKS2, which we named GmMKS2
A BLASTN search using the predicted GmMKS2 genomic sequence as a query against the RefSeq RNA database at NCBI of G. max identified two transcript variants potentially transcribed from this locus: variant X1 and variant X2

Summary

Introduction

The formation of acyl carrier protein (ACP) thioesters of acyl groups containing between2 and 16 carbon atoms occurs in fatty acid (FA) biosynthesis and is catalyzed by a set of fatty acid synthase (FAS) enzymes highly conserved across various species [1,2]. The formation of acyl carrier protein (ACP) thioesters of acyl groups containing between. Plants contain general and lineage-specific acyl-ACP thioesterases (acyl-ACP TEs) that hydrolyze acyl-ACPs of specific length and structure to generate free fatty acids that are used in further metabolism [3,4]. Plants have thioesterases (TEs) that use fatty acyl-CoA substrates (which occur, for example, during fatty acid degradation) that serve to generate free fatty acids. All known fatty acyl-TEs have been grouped into 23 different families according to their amino acid sequence, but on the basis of the tertiary structure, they mainly fall into two types of folds: the α/β-hydrolase fold superfamily and the hotdog fold superfamily [5].

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